Crystal structure of the Golgi casein kinase

Abstract

The family with sequence similarity 20 (Fam20) kinases phosphorylate extracellular substrates and play important roles in biomineralization. Fam20C is the Golgi casein kinase that phosphorylates secretory pathway proteins within Ser-x-Glu/pSer motifs. Mutations in Fam20C cause Raine syndrome, an osteosclerotic bone dysplasia. Here we report the crystal structure of the Fam20C ortholog from Caenorhabditis elegans. The nucleotide-free and Mn/ADP-bound structures unveil an atypical protein kinase-like fold and highlight residues critical for activity. The position of the regulatory αC helix and the lack of an activation loop indicate an architecture primed for efficient catalysis. Furthermore, several distinct elements, including the presence of disulfide bonds, suggest that the Fam20 family diverged early in the evolution of the protein kinase superfamily. Our results reinforce the structural diversity of protein kinases and have important implications for patients with disorders of biomineralization.

Keywords: Fam20A; Fam20B; amelogenesis imperfecta; enamel renal syndrome; hypophosphatemia.

Fig. 1.

C. elegans Fam20 exhibits a protein kinase-like fold contained in a shell-like structure. (A) Ribbon representation of ceFam20. The N- and C-lobes are shown in magenta and teal, respectively. The α6 helix (PKA αC equivalent) is highlighted in orange. The N-terminal segment and the insertion domain are in white and pink, respectively. (B) The amino acid sequence of ceFam20 is shown depicting the secondary structural elements, color-coded as in A. The disulfide bonds are indicated with green lines, and the N-linked glycosylation sites are highlighted in red.

Fig. 2.

Fam20C activity and specificity are evolutionarily conserved. (A) Human Fam20C and ceFam20 phosphorylated the Fam20C model peptide substrate β28–40, whereas Fam20B did not. (B) Fam20B phosphorylated a model substrate representing the tetrasaccharide core linkage region in proteoglycans (GlcAβ1–3Galβ1–3Galβ1–4Xylβ1-O-benzyl), whereas Fam20C and ceFam20 did not. (C) Peptides containing variants of the Ser-x-Glu Fam20C consensus motif were synthesized to assess the specificity of ceFam20. The Ser was changed to Thr or Tyr, and the n+2 residue was changed to Asp. Incorporation of phosphate was monitored by autoradiography.

Fig. 3.

The Mn/ADP-bound structure of ceFam20 reveals critical residues required for catalysis. (A) Enlarged image of the nucleotide-binding pocket showing the detailed molecular interactions important for catalysis. The ADP molecule and the two Mn²⁺ ions are shown as sticks and spheres, respectively. Salt bridge and hydrogen bond interactions are shown as dashed lines. (B) Human Fam20C mutants display reduced kinase activity. Mutant Fam20C proteins were assayed against β28–40 peptide, and their activity relative to that of the WT enzyme is depicted graphically. The amino acids in brackets indicate the corresponding residues in ceFam20.

Fig. 4.

Insights into substrate specificity. (A) Structural representation of the active site of ceFam20, highlighting residues potentially involved in substrate recognition. The Mn/ADP, the catalytic Asp (D366), and three Fam20C-specific basic residues (R314, K319, and K320) are shown. (B) Sequence alignment of the β8-α9 loop within the Fam20 family members. The three Fam20C-specific basic residues are highlighted.

Fig. 5.

ceFam20 has unique spine structures. The N-lobe, C-lobe, and αC helix of PKA are colored in magenta, teal, and orange, respectively. Corresponding regions in ceFam20 are highlighted in the same color scheme. The regulatory and catalytic spines are depicted in red and green, respectively. Residues involved in the formation of the spines are indicated.

Fig. 6.

Structure modeling provides insights into Fam20C-related human disorders. The homology structure model of human Fam20C is shown as a ribbon diagram. The model was generated using Swiss-Model (44). Disease mutations are highlighted in red.